Using tools from the fields of inorganic chemistry, biochemistry and molecular biology we are attempting to understand the mechanism of mercury responsive gene expression at the molecular level. The central component of a mercury-responsive genetic switch, the MerR, protein recently became available in large quantities, however very little is known about its physical or biological properties, and much less about the way the metal or the protein might effect gene regulation. MerR is an essential member of bacterial mercury detoxification systems where its role is to activate mercury-specific cellular defenses when subtoxic quantities of mercury are present. This gene-regulatory, DNA-binding protein interacts specifically with the control regions of the mercury resistance operon and exerts positive and negative control over gene expression in response to mercuric ion. Our efforts to characterize this mechanism, particularly the "metal sensing apparatus" (an inorganic ligand-receptor interaction) fall into two categories: chemical studies of protein- metal interactions and studies of the biological activity of the protein in combination with the cellular transcription apparatus. The former group of experiments involves thermodynamic studies and molecular probes of the metal-protein interactions. Both novel and well established techniques will be used to explore this small protein and its interactions with metals and cellular transcription apparatus. 199Hg NMR will be developed as a molecular probe of metal-biopolymer interactions and will be applied to help elucidate the mechanism utilized by the MerR protein. A recently developed, high resolution footprinting technique based on Fenton chemistry will be used alongside traditional interference and protection methods to map the specific DNA sights which interact with the principle components of this genetic switch: MerR protein, RNA polymerase and the DNA fragment containing the mer operator and promoter. Specific assays measuring transcription activation by MerR are also planned. Elucidation of mercury-responsive gene regulation in the MerR system may provide a molecular paradigm for metal- responsive gene regulation, which is the core of several eukaryotic and prokaryotic heavy-metal homeostasis systems.